Method for production of molded bodies, in particular optical structures and use thereof

ABSTRACT

The present invention relates to a method for manufacture a body from a thermoplastic plastic with a three-dimensionally structured surface, wherein molding is performed directly from a master made of glass coated with metal oxide, without deposition of further coatings on a surface of the master. The invention also relates to bodies manufactured with this method from a thermoplastic featuring a three-dimensionally structured surface, as well as to planar optical structures likewise manufactured with this method for generating evanescent-field measuring platforms and to a use thereof. The invention furthermore relates to a planar optical structure for generating an evanescent-field measuring platform, comprising a first essentially optical transparent, waveguiding layer (a) with refractive index n 1  and a second essentially optical transparent layer (b) with refractive index n 2 , where n 1 &gt;n 2 , in a case of an embodiment of a planar optical film waveguide, or comprising a metal layer (a′) and a second layer (b), in a case of an embodiment for generating a surface plasmon resonance, wherein the second layer (b) comprises a material from a group comprising cyclo-olefin polymers and cyclo-olefin copolymers.

BACKGROUND OF THE INVENTION

The invention described herein comprises a novel process formanufacturing a body from a thermoplastic plastic with athree-dimensionally structured surface, wherein molding is performeddirectly from a master made of glass coated with metal oxide, withoutdeposition of further coatings on a surface of the master. A methodaccording to the invention thus comprises fewer operational steps thancorresponding conventional molding processes, which will lead todecreases in manufacturing costs. Furthermore, as a result of a smallernumber of processing steps to be performed before molding on acorresponding master, risk of damage to the surface of the master, whichis inevitably carried over as defects in molded bodies, is markedlyreduced, which means a substantial advance in a production process.

A surface free of defects is especially important for optimization ofplanar waveguides, especially for applications in bioanalytics, in orderto avoid scatter of guided excitation light at scatter centers and/or asa result of high surface roughness. A goal is to achieve a lowestpossible surface roughness of a planar waveguide.

If in-coupling of excitation light into a waveguide takes place by useof a diffractive relief grating, then an extraordinary uniformity andreproducibility of these structures with dimensions of often only a fewnanometers is necessary. In a case of manufacture of such waveguidesfrom plastic substrates, high requirements are thus placed oncorresponding molding processes.

Such periodic structures like surface relief gratings, in combinationwith thin metal layers deposited thereon (typically layers of gold orsilver with thicknesses on the order of magnitude of about 40 nm-200 nm)on an underlying dielectric layer with a lower refractive index are alsosuitable for creating conditions for a surface plasmon resonance which,similar to waveguiding in an optical waveguide, is associated withformation of an evanescent field (with exponential decay in intensity ina direction of adjacent media), along propagation of the surface plasmon(instead of a guided wave). The formation of the evanescent field isexplained more precisely below in an example of an optical waveguide.The term waveguiding is understood here to mean that a propagationlength of a wave “guided” in a highly refractive layer shall correspond,expressed in a ray model of classical optics, at least to a distance inthis layer between two total reflections at interfaces of this layer toadjacent low-refractive media or layers, opposite to one another. In alow-loss waveguide, propagation length may amount to several centimeters(or even kilometers, as in telecommunications); in a waveguide with alarge-area modulated grating structure (depending on depth of grating)it may also measure some micrometers to a few millimeters, which iscomparable with a typical propagation length of surface plasmons(typically on the order of magnitude of 100 μm). Such essentially planaroptical structures, such as optical film waveguides and structures witha thin metal coating on a dielectric substrate of lower refractiveindex, which are described more closely below and which are suitable forgenerating an evanescent field, should be commonly described as “planaroptical structures for generating evanescent field measuring platforms”.

The invention also relates to variable embodiments of “planar opticalstructures for generating evanescent-field measuring platforms”, whereina layer (b) comprises a material from a group comprising cyclo-olefinpolymers and cyclo-olefin copolymers. In particular the inventionrelates to a planar optical film waveguide, comprising a firstessentially optically transparent waveguiding layer (a) with refractiveindex n₁ and a second essentially optically transparent layer (b) withrefractive index n₂, where n₁>n₂, wherein the second layer (b) comprisesa material from a group formed by cyclo-olefin polymers and cyclo-olefincopolymers.

The invention relates also to an analytical system with a planar opticalstructure according to the invention for generating an evanescent fieldmeasurement arrangement as a main component, as well as methods formanufacturing such planar optical structures and methods based on usethereof for detecting one or more analytes in one or more samples.

To achieve lower limits of detection, numerous measurement arrangementshave been developed in the last years, in which detection of an analyteis based on its interaction with an evanescent field, which isassociated with light guiding in an optical waveguide, whereinbiochemical or biological recognition elements for specific recognitionand binding of analyte molecules are immobilized on a surface of thewaveguide.

When a light wave is in-coupled into an optical waveguide surrounded byoptically rarer media, i.e. media of a lower refractive index, the lightwave is guided by total reflection at interfaces of a waveguiding layer.In this arrangement, a fraction of electromagnetic energy penetratesinto the optically rarer media. This portion is termed an evanescent ordecaying field. A strength of the evanescent field depends to a verygreat extent on a thickness of the waveguiding layer itself and on aratio of refractive indices of the waveguiding layer and surroundingmedia. In the case of thin-film waveguides, i.e. layer thicknesses thatare the same as or thinner than a wavelength of light to be guided,discrete modes of guided light can be distinguished. Analyte detectionmethods in an evanescent field have an advantage in that interactionwith an analyte is limited to a penetration depth of the evanescentfield into an adjacent medium, on the order of magnitude of some hundrednanometers, and interfering signals from a depth of the medium can belargely avoided. First proposed measurement arrangements of this typewere based on highly multi-modal, self-supporting single-layerwaveguides, such as fibers or plates of transparent plastic or glass,with thicknesses from some hundred micrometers up to severalmillimeters.

Planar thin-film waveguides have been proposed in order to improvesensitivity and at the same time facilitate mass production. A planarthin-film waveguide in a simplest case comprises a three-layer system:carrier material, waveguiding layer, and superstrate (i.e. a sample tobe analyzed), wherein the waveguiding layer has a highest refractiveindex. Additional intermediate layers can further improve action of theplanar waveguide. Essential requirements placed on properties of thewaveguiding layer itself and on a layer in contact therewith in adirection of the substrate or carrier material or on the substrate orthe carrier material itself are in this case a maximum possibletransparency at a wavelength of light to be guided, together with aminimum possible intrinsic fluorescence and a minimum possible surfaceroughness, in order for the light to be guided as free from interferenceas possible. Suitable substrate materials are therefore, for example,glass or plastics with corresponding properties, as has been widelydescribed (e.g. in WO 95/33197 and WO 95/33198), with glass havingproved more advantageous to date with regard to poverty of fluorescence(on excitation in a visible spectrum) and low surface roughness. Areason for the low surface roughness which can be achieved for glasssubstrates is in particular a possibility of heating these up to hightemperatures so that formation of a roughness-enhancing microcolumnstructure can be largely prevented.

In the case of plastic substrates, deposition of an intermediate layerbetween a substrate and a waveguiding layer is often necessary e.g. inorder for contribution of the substrate's intrinsic fluorescence to bereduced for fluorescence measurements.

An optical waveguide with a substrate of plastic or a high organicportion and with an inorganic waveguiding layer, as well as methods formanufacture of this waveguide, are described in EP 533,074.Thermoplastically processable plastics, in particular polycarbonates,polymethylmethacrylates (PMMA) and polyesters, are preferred here.

Within this group of plastics, PMMA is known for having best opticalproperties, i.e. in particular poverty of fluorescence. A disadvantageof PMMA that has been described, however, is its low temperaturestability, which does not permit continuous operating temperatures above60° C. to 90° C., as required in some cases e.g. for nucleicacid-hybridization assays.

Less favorable physicochemical, especially optical, properties of knownfilm waveguides comprising plastics as substrate (=essentially opticallytransparent layer (b)), contrasts with easier processability of thesesubstances versus glass substrates, especially for producing astructured surface, e.g. through molding of a suitably structuredmaster. Such molding processes for producing structured plastic surfacesgenerally cost less than a usual photolithographic surface structuringof glass substrates.

There is thus a need for optical waveguides, or general opticalstructures for generating an evanescent field measuring platform, whichhave similarly favorable optical properties, such as waveguides based onglass substrates, but which can be produced at lower cost.

Surprisingly it has now been found that, by using substrates ofcyclo-olefin polymers (COP) or cyclo-olefin copolymers (COC), which arenot mentioned in EP 533,074, it is possible to manufacture opticalstructures for generating evanescent-field measuring platforms andespecially film waveguides, which are characterized by especially lowintrinsic luminescence or fluorescence, with this being of greatadvantage in particular for fluorescence-based measuring methods, andwhich also show very low propagation losses of guided light. A newmethod was also surprisingly found for manufacturing film waveguidesaccording to the invention by means of which these can be moldedespecially easily and in very good quality from a master.

Some favorable properties of optical components based on COP, comparedwith other plastics used in optics, which are listed in a productbrochure of Nippon Zeon Co. Ltd., under the heading “Zeonex”, includevery low water absorption, high heat resistance, low content ofimpurities and relatively good chemical resistance.

In U.S. Pat. No. 6,063,886, various cyclo-olefin copolymers andcomponents manufactured therefrom by injection molding are claimed,especially for optics. However, there are no references to their use foroptical waveguides with associated highly specific requirements. Also noinformation at all is given to indicate possible molding processes forgeneration of three-dimensional structures of COC.

In U.S. Pat. No. 6,120,870, an optical “disk”, based on COP, and amolding process for manufacturing this (structured) disk from a siliconmaster are described, wherein a resin layer between the master and a COPdisk to be structured is used in each of specified variants of a moldingprocess, with this layer being cured either photochemically, by UVlight, or thermally.

In U.S. Pat. No. 5,910,287, microtiter plates (“multi-well plates”) aredescribed in which COC or COP is used as material for a floor of wellsin order to reduce intrinsic fluorescence of such a plate forfluorescence-based tests. Manufacturing processes described includereaction injection molding (RIM) and liquid injection molding (LIM).

RIM (reaction injection molding) is a low-pressure mixing and injectionprocess in which two or more liquid components are injected into aclosed mold, where a plastic body is formed during rapid polymerization.Possible problems of an RIM process that have been described are inparticular blister formation during a curing process, poor mold fillingand difficult demoldability of a manufactured plastic body (H. Vollmer,W. Ehrfeld, P. Hagmann, “Untersuchungen zur Herstellung vongalvanisierbaren Mikrostrukturen mit extremer Strukturhöhe durchAbformung mit Kunststoff im Vakuum-Reaktionsgiessverfahren”, Report 4267of the KfK, Karlsruhe, Germany, 1987; P. Hagmann, W. Ehrfeld,“Fabrication of Microstructures of extreme Structural Heights byReaction Injection Molding”, International Polymer Processing IV (1989)3, pp. 188-195). Even if these problems are largely solved, adisadvantage of the RIM process which remains is a relatively long cycletime of several minutes (T. Bouillon, “Mikromechanik—Bedeutung undAnwendung von Mikrostrukturen aus Kunststoffen, Metallen und Keramik”,study paper at the IKV, RWTH Aachen, cited in A. Rogalla, “Analyse desSpritzgiessens mikrostrukturierter Bauteile aus Thermoplasten”, IKVBerichte aus der Kunststoffverarbeitung, Vol. 76, Verlag Mainz,Wissenschaftsverlag Aachen, Germany, 1998). An LIM process (liquidinjection molding) is described as even more time-consuming, withtypical cycle times of 5 to 10 minutes.

In contrast to the molding processes described above, a variotherminjection process (A. Rogalla, “Analyse des Spritzgiessensmikrostrukturierter Bauteile aus Thermoplasten”, IKV Berichte aus derKunststoffverarbeitung, Vol. 76, Verlag Mainz, WissenschaftsverlagAachen, Germany, 1998) is preferred for the process according to theinvention in order to mold from a master of glass coated with a metaloxide, as part of a molding tool, and to manufacture a planar opticalfilm waveguide. This purely physical process, based on liquefying atelevated temperature of plastic initially provided as pellets, enablesplastic bodies to be manufactured with even very fine structures in veryshort cycle times (W. Michaeli, H. Greif, G. Kretzschmar, H. Kaufmannand R. Bertulait, “Technologie des Spritzgiessens”, Carl Hanser VerlagMunich Vienna 1993, p. 69).

DETAILED DESCRIPTION OF THE INVENTION

A first subject of the invention is a method for manufacturing a bodyfrom a thermoplastic plastic with a three-dimensionally structuredsurface, wherein molding is performed directly from a master made ofglass coated with metal oxide, without deposition of further coatings ona surface of the master.

This molding method according to the invention offers a number ofadvantages over known processes for molding from coated, e.g.galvanized, masters, for example from so-called nickel shims. Inparticular, materials to be molded from, according to the invention,i.e. glass coated with metal oxide, are harder and morescratch-resistant, thus allowing a larger number of moldings to bemanufactured from one and the same master. Moreover, additional processsteps, such as deposition of an additional coating, are avoided whenpreparing the master. This not only simplifies preparation of themaster, but also avoids a risk of generating additional defects on themaster as a result of additional processing steps that are otherwiseneeded. Thus, the master is prepared without depositing further coatingsprior to molding.

It is preferred if the metal oxide of the master is a material from agroup of materials comprising TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂,with particular preference being for TiO₂, Ta₂O₅ or Nb₂O₅.

The master itself may be manufactured using standard methods formicrostructuring, such as photolithograpy, laser ablation, electron beamor ion beam processing.

Thereby, the process is characterized in that three-dimensionalstructures measuring 1-1000 nm and 1 μm to 1000 μm are enabled of beingmolded in a single molding step. In particular, if very small and alsorelatively large structures are molded at the same time, requirementsregarding surface quality of the master are of course very high. Ahigher cost in manufacture of the master, however, is more than offsetby saving of a second process step for a molded product which wouldotherwise be needed.

Furthermore, the method according to the invention allows extendedbodies with a three-dimensionally structured surface of more than 1 cm²,preferably of more than 10 cm², especially preferably of more than 100cm², to be molded in a single step. For example, bodies of a size of astandard microtiter plate, with surface structures of nanometer ordersof magnitude, can be molded simultaneously.

Molding based on the method according to the invention may be performedusing all known processes (such as RIM, LIM and the like) which arecompatible with properties of the master (e.g. in respect of resistanceto high temperatures or pressures). For manufacture of a small productseries, hot embossing of plastics is often used. Accordingly, it ispreferred if molding is performed using a method from a group ofprocesses comprising injection molding, reaction injection molding(RIM), liquid injection molding (LIM) and hot embossing.

Molding by use of an injection molding process is especially preferred,and a variotherm injection molding process is most particularlypreferred.

It is preferred moreover if a molding material used in the methodaccording to the invention for production of a body with athree-dimensionally structured surface includes a material from a groupcomprising polycarbonates, polymethylmethacrylates, cyclo-olefinpolymers and cyclo-olefin copolymers, where a group formed bycyclo-olefin polymers and cyclo-olefin copolymers is especiallypreferred.

Suitable cyclo-olefins are described in U.S. Pat. No. 5,278,238 (B. L.Lee et al.), U.S. Pat. No. 4,874,808 (Minami et al.), U.S. Pat. No.4,918,133 (Moriya et al.), U.S. Pat. No. 4,935,475 (Kishiinura et al.),U.S. Pat. No. 4,948,856 (Minchak et al.), U.S. Pat. No. 5,115,052(Wamura et al.), U.S. Pat. No. 5,206,306 (Shen), U.S. Pat. No. 5,270,393(Sagane et al.), U.S. Pat. No. 5,272,235 (Wakatsuru et al.), U.S. Pat.No. 5,278,214 (Moriya et al.), U.S. Pat. No. 5,534,606 (Bennett et al.),U.S. Pat. No. 5,532,030 (Hirose et al.), U.S. Pat. No. 4,689,380 (Hiroseet al.), U.S. Pat. No. 4,689,380 (Nahm et al.) and U.S. Pat. No.4,899,005 (Lane et al.). Cyclo-olefins (e.g. cyclopentene, cyclohexeneand cycloheptene) and polyethylene copolymers thereof are preferred, asare corresponding thermoplastic olefin polymers of amorphous structure(TOPAS line) from Hoechst (Germany). Of these, plastics TOPAS 8007,5013, 6013, 6015 and 6017 are especially preferred. Cyclo-olefinpolymers sold by the company Nippon Zeon Co., Japan, under the productname ZEONEX (e.g. polymers 480, 480R, E48R and 490K) and ZEONOR(polymers 1020R, 1060R, 1420R, 1600R) are also preferred.

A subject of the invention is in particular a method for manufacture ofa planar optical structure for generating an evanescent-field measuringplatform, wherein the evanescent-field measuring platform comprises amultilayer system, with a metal layer (a′) or an essentially opticallytransparent, waveguiding layer (a) with refractive index n₁ and at leasta second, essentially optically transparent layer (b) with refractiveindex n₂, where n₁ 22 n₂, and where the second layer (b) comprises athermoplastic plastic and is molded directly from a master made of glasscoated with a metal oxide, as part of a molding tool, without depositionof further coatings on a surface of the master.

A preferred variant here is a method for manufacture of a planar opticalstructure for generating an evanescent-field measuring platform, whereinthe evanescent-field measuring platform is a planar optical structurefor generating a surface plasmon resonance. The metal layer of thisoptical structure preferably comprises gold or silver. Especiallysuitable in this case are layer thicknesses between 40 nm and 200 nm,with thicknesses between 40 nm and 100 nm being especially preferred. Itis an advantage if material of layer (b) or of an optional additionaldielectric layer (buffer layer) which is in contact with the metal layerhas a low refractive index n<1.5, especially preferably n<1.35.

In another, especially preferred variant, a manufacturing processaccording to the invention is a method for manufacture of a planaroptical waveguide, comprising a first essentially optically transparent,waveguiding layer (a) with refractive index n₁ and a second essentiallyoptically transparent layer (b) with refractive index n₂, where n₁>n₂,and where the second layer (b) of the waveguide comprises athermoplastic plastic and is molded directly from a master made of glasscoated with a metal oxide, as part of a molding tool, without depositionof further coatings on a surface of the master.

The term “planar” is understood to mean here that, apart from surfaceroughness or structuring for light in-coupling or out-coupling and,where applicable, recesses structured in a surface of the waveguide forcreation of sample compartments, a radius of curvature of the surface ofthe waveguide both parallel with and perpendicular to a direction ofpropagation of light during waveguiding is at least 1 cm, preferably atleast 5 cm.

The term “essentially optically transparent” is understood to mean thata layer thus characterized is a minimum of 95% transparent at least at awavelength of light delivered from an external light source for itsoptical path perpendicular to the layer, provided the layer is notreflecting. In a case of partially reflecting layers, “essentiallyoptically transparent” is understood to mean that a sum of transmittedand reflected light and, if applicable, light in-coupled into a layerand guided therein amounts to a minimum of 95% of delivered light at apoint of incidence of the delivered light.

For the method according to the invention for manufacture of a planaroptical structure for generating an evanescent-field measuring platformand, in particular also for generating a planar optical film waveguide,the same preferences apply as those stipulated above in general for themethod for manufacturing a body made of a thermoplastic plastic with athree-dimensionally structured surface.

The process comprises grating structures (c) or (c′) located on thesurface of the master and formed as relief gratings being transferred toa surface of layer (b) during a molding step. This thus means that thegrating structures (c) and/or (c′) formed as relief gratings aregenerated in a surface of layer (b) by molding from a master withsurface relief gratings complementary to grating structures (c) and/or(c′), respectively.

The manufacturing process according to the invention also allows raisedareas formed on the surface of the master to be formed during themolding step as recesses in layer (b). The recesses in layer (b)preferably have a thickness of 20 μm to 500 μm, preferably 50 μm to 300μm.

In particular, it is characteristic for the manufacturing processaccording to the invention that grating structures (c) and/or (c′) asrelief gratings with a depth of 3 nm to 100 nm, preferably of 10 nm to30 nm, and recesses with a depth of 20 μm to 500 μm, preferably of 50 μmto 300 μm, can be molded simultaneously in a single step.

It is preferred if material used in the process according to theinvention for generating the essentially transparent layer (b) of theplanar optical structure for generating an evanescent-field measuringplatform includes a material from a group comprising polycarbonates,polymethylmethacrylates, cyclo-olefin polymers and cyclo-olefincopolymers. Especially preferred is a material from a group formed bycyclo-olefin polymers and cyclo-olefin copolymers.

A further subject of the invention is a body made of a thermoplasticplastic with a three-dimensionally structured surface wherein molding ofthe structured surface is performed directly from a master made of glasscoated with metal oxide, without deposition of further coatings on asurface of the master, in a manufacturing process according to theinvention as defined in one of the embodiments.

A molded surface of the body may have structures with dimensions of 1nm-1000 nm or also of 1 μm-1000 μm. In particular, the molded surfacemay comprise structures with dimensions of 1 nm-1000 nm and of 1 μm to1000 μm, which are molded in a single step.

The body according to the invention may have an extendedthree-dimensionally structured surface of more than 1 cm², preferably ofmore than 10 cm², especially preferably of more than 100 cm², which ismolded in a single step.

It is preferred if molding of the body is performed using a method froma group of processes comprising injection molding, reaction injectionmolding (RIM), liquid injection molding (LIM) and hot embossing.Especially preferred is an injection molding process, most especiallypreferred is a variotherm injection molding process.

It is further preferred if molding material used in the processaccording to the invention, for producing a body with athree-dimensionally structured surface, includes a material from a groupcomprising polycarbonates, polymethylmethacrylates, cyclo-olefinpolymers and cyclo-olefin copolymers. In this case, a material from agroup comprising cyclo-olefin polymers and cyclo-olefin copolymers isespecially preferred.

A subject of the invention is also a planar optical structure forgenerating an evanescent-field measuring platform, wherein theevanescent-field measuring platform comprises a multilayer system, witha metal layer and/or an essentially optically transparent waveguidinglayer (a) with refractive index n₁ and at least a second, essentiallyoptically transparent layer (b) with refractive index n₂, where n₁>n₂,and where the second layer (b) comprises a thermoplastic plastic and ismolded directly from a master made of glass coated with a metal oxide,as part of a molding tool, without deposition of further coatings on asurface of the master, in a manufacturing method according to theinvention as described in one of the embodiments.

In a preferred variant of a planar optical structure according to theinvention for generating an evanescent-field measuring platform, this isa planar optical structure for generating a surface plasmon resonance.This planar optical structure preferably comprises a metal layer of goldor silver.

Especially preferred, thereby, are metal layer thicknesses between 40 nmand 200 nm, with thicknesses between 40 nm and 100 nm being especiallypreferred. It is an advantage if a material of layer (b) or of anoptional additional dielectric layer (buffer layer) which is in contactwith the metal layer has a low refractive index n<1.5, especiallypreferably n<1.35.

In another, especially preferred embodiment of a planar opticalstructure according to the invention for generating an evanescent-fieldmeasuring platform, the structure is a planar optical film waveguide,comprising a first essentially optically transparent, waveguiding layer(a) with refractive index n₁ and a second essentially opticallytransparent layer (b) with refractive index n₂, where n₁>n₂, and wherethe second layer (b) of the film waveguide comprises a thermoplasticplastic and is molded directly from a master made of glass coated withmetal oxide, as part of a molding tool, without deposition of furthercoatings on a surface of the master, in a manufacturing method accordingto the invention as described in one of the embodiments.

It is characteristic for the planar optical structure according to theinvention for generating an evanescent-field measuring platform thatgrating structures (c) or (c′) located on the surface of the master andformed as relief gratings are transferred to a surface of layer (b)during a molding step. This means in particular that the gratingstructures (c) and/or (c′) formed as relief gratings are generated in asurface of layer (b) by molding from a master with surface reliefgratings complementary to grating structures (c) and/or (c′),respectively.

A characteristic of a special embodiment of the planar optical structurefor generating an evanescent-field measuring platform according to theinvention is that raised areas formed on the surface of the master aremolded as recesses in layer (b) during the molding step. The recesses inlayer (b) preferably have a depth of 20 μm to 500 μm, especiallypreferably 50 μm to 300 μm.

It is also preferred if the planar optical structure according to theinvention for generating an evanescent-field measuring platform has anextended three-dimensionally structured surface of more than 1 cm²,preferably of more than 10 cm², especially preferably of more than 100cm², which is molded in a single molding step.

The essentially optically transparent layer (b) of a planar opticalstructure according to the invention for generating an evanescent-fieldmeasuring platform may be molded using a method from the group ofprocesses comprising injection molding, reaction injection molding(RIM), liquid injection molding (LIM) and hot embossing. Molding ispreferably performed using an injection molding process, especiallypreferably using a variotherm injection molding process.

It is an advantage if material of the second essentially opticallytransparent layer (b) of the planar optical structure according to theinvention for generating an evanescent-field measuring platform, as usedin the manufacturing process according to the invention, comprises amaterial from a group comprising polycarbonates,polymethylmethacrylates, cyclo-olefin polymers and cyclo-olefincopolymers, with material from a group comprising cyclo-olefin polymersand cyclo-olefin copolymers being especially preferred.

Planar optical structures from multilayer systems, for generating anevanescent-field measuring platform, and especially planar optical filmwaveguides with an essentially optically transparent layer (b) ofcyclo-olefin polymers or cyclo-olefin copolymers are not known in theprior art, as stated hereinbefore.

A further subject of the invention is therefore, regardless also of themanufacturing process, a planar optical structure for generating anevanescent-field measuring platform, wherein the evanescent-fieldmeasuring platform comprises a multilayer system, with a metal layerand/or an essentially optically transparent, waveguiding layer (a) withrefractive index n₁ and at least a second, essentially opticallytransparent layer (b) with refractive index n₂, where n₁>n₂, and wherethe second layer (b) consists of a thermoplastic plastic and comprises amaterial from a group comprising cyclo-olefin polymers and cyclo-olefincopolymers.

In a preferred embodiment, this structure is a planar optical structurefor generating a surface plasmon resonance. This planar opticalstructure preferably comprises a metal layer of gold or silver.

Another preferred embodiment of a planar optical structure according tothe invention for generating an evanescent-field measuring platformcharacterized in that the evanescent-field measuring platform is aplanar optical film waveguide, comprises a first essentially opticallytransparent waveguiding layer (a) with refractive index n₁ and a secondessentially optically transparent layer (b) with refractive index n₂,where n₁>n₂, and where the second layer (b) of the film waveguidecomprises a material from a group formed by cyclo-olefin polymers andcyclo-olefin copolymers.

It is further preferred if a refractive index of the first opticallytransparent layer (a) is greater than 1.8. Numerous materials aresuitable for optical layer (a). Without loss of generality, it ispreferred if the first optically transparent layer (a) is a materialfrom a group comprising TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂, withspecial preference being for TiO₂ or Nb₂O₅ or Ta₂O₅.

Light delivered from an external light source in a direction of layer(a) (or layer (a′), respectively), i.e. both light irradiated throughlayer (b) in the direction of layer (a) (or the metal layer,respectively) and also light irradiated from an opposite side, ifnecessary through a medium located above layer (a) (or the metal layer,respectively), in the direction of layer (a) (or the metal layer,respectively), shall be called generally according to the presentinvention an “excitation light”. This excitation light may serve bothfor excitation of luminescence, or, more specifically, fluorescence orphosphorescence, and also for Raman radiation of molecules adjacent tolayer (a) (or to the metal layer, respectively) or also for in-couplinginto layer (a), for determination of actual coupling parameters, such asan in-coupling angle, or for excitation of a surface plasmon in a metallayer, for determination of a resonance angle for surface plasmonresonance, or also other parameters, such as a phase difference oflight, between a split beam of excitation light passing through a regionprovided on layer (a) for detection of at least one analyte and anothersplit beam passing through a referencing region, in an interferometricmeasurement arrangement.

It is also preferred if the waveguiding layer (a) of a planar opticalfilm waveguide according to the invention, as a preferred embodiment ofa planar optical structure according to the invention for generating anevanescent-field measuring platform, is in optical contact with at leastone optical coupling element for in-coupling of excitation light of atleast one wavelength from at least one light source into layer (a).

Several methods are known for coupling excitation light into a planarwaveguide. Earliest methods used were based on butt-end coupling orprism coupling, wherein generally a liquid is introduced between a prismand a waveguide to reduce reflections resulting from air gaps. These twomethods are mainly suitable in combination with waveguides havingrelatively large layer thickness—i.e. especially self-supportingwaveguides—and a refractive index significantly below 2. By contrast,for coupling of excitation light into very thin waveguiding layers ofhigh refractive index, use of coupling gratings is a substantially moreelegant method.

It is preferred if, for in-coupling of excitation light into theoptically transparent layer (a), this layer is in optical contact withat least one optical in-coupling element from a group comprising prismcouplers, evanescent couplers with combined optical waveguides withoverlapping evanescent fields, butt-end couplers with focusing lenses,preferably cylinder lenses, arranged in front of one face of awaveguiding layer, and grating couplers.

It is especially preferred if the excitation light is in-coupled intothe optically transparent layer (a) using at least one grating structure(c) which is featured in the optically transparent layer (a).

Out-coupling of light guided in layer (a) may in principle take placevia the same kind of optical coupling elements as those namedhereinbefore for in-coupling. It is preferred if light guided inoptically transparent layer (a) is out-coupled using grating structures(c′), which are featured in the optically transparent layer (a).

Grating structures (c) and (c′) featured in the optically transparentlayer (a) may have the same or different periods and be arrangedparallel or not parallel with each other. In general, grating structures(c) and (c′) can be used alternately as in-coupling and/or out-couplinggratings.

Apart from the refractive index of the waveguiding optically transparentlayer (a), a thickness thereof is a second decisive parameter forgenerating both a strongest possible evanescent field at interfaces withadjacent layers which have a lower refractive index, and a highestpossible energy density within layer (a). Thereby, intensity of theevanescent field increases with decreasing thickness of the waveguidinglayer (a) as long as the thickness of the layer is sufficient to guideat least one mode of an excitation wavelength. Thereby, a minimum“cut-off” thickness for guiding a mode is dependent on a wavelength oflight to be guided. It is greater for longer wavelength light than forshort wavelength light. As the layer thickness approaches this “cut-off”point, however, adverse propagation losses also show a marked increase,which additionally exerts a downward limit on a preferred layerthickness. Preference is for thicknesses of optically transparent layer(a) which allow only 1 to 3 modes of a specified excitation wavelengthto be guided, with very particular preference being for layerthicknesses which lead to monomodal waveguides for this excitationwavelength. It is clear in this case that a discrete modal character ofguided light only relates to transversal modes.

These requirements lead to a product from the thickness of layer (a) andits refractive index advantageously amounting to one-tenth to one whole,preferably one-third to two-thirds, of a wavelength of an excitationlight in-coupled into layer (a).

It is also preferred if the grating structures (c) and/or (c′) arerelief gratings with a grating structure depth of 3 to 100 nm,especially preferably of 10 to 30 nm. It is an advantage if a ratio ofmodulation depth to thickness of the first optically transparent layer(a) is equal to or less than 0.2.

A level of propagation losses of a mode guided in an opticallywaveguiding layer (a) is determined to a large extent by surfaceroughness of an underlying carrier layer and by absorption throughchromophores that may be present in this carrier layer, which inaddition carries a risk that luminescence, which is unwanted for manyapplications, may be excited in this carrier layer through penetrationof an evanescent field of a mode guided in layer (a). Thermal tensionmay also occur owing to different thermal expansion coefficients of theoptically transparent layers (a) and (b). It may therefore be anadvantage if an additional optically transparent layer (b′) with a lowerrefractive index than that of layer (a), and with a thickness of 5nm-10,000 nm, preferably 10 nm-1000 nm, is located between the opticallytransparent layers (a) and (b) and is in contact with layer (a). Thisintermediate layer has a function of reducing surface roughness belowlayer (a) or reducing penetration of an evanescent field of light guidedin layer (a) into at least one underlying layer, or improving adhesionof layer (a) on the at least one underlying layer, or reducing thermallyinduced tension within a film waveguide, or chemically isolating theoptically transparent layer (a) from underlying layers by virtue ofsealing micropores in layer (a) against underlying layers.

The following preferences apply in turn not only for a planar opticalfilm waveguide according to the invention, but also for a more generalsubject of the invention of a planar optical structure, comprising amultilayer system, for generating an evanescent-field measuringplatform.

Special embodiments of the planar optical structure according to theinvention for generating an evanescent-field measuring platform compriselarge-area grating structures (c) and/or (c′) that cover extensivesurface areas of the optical structure, preferably an entire surfacearea thereof. The planar optical structure according to the inventionfor generating an evanescent-field measuring platform may also featuremultiple grating structures (c) and/or (c′) on a common, continuoussubstrate in the essentially optically transparent layer (a) and/or themetal layer.

For example, for in-coupling of excitation light of differentwavelengths, such an embodiment may be advantageous which comprises asuperposition of at least two grating structures of differingperiodicity with grating lines arranged parallel or not parallel withone another.

A characteristic for another preferred embodiment is at least onegrating structure (c) and/or (c′) which shows a three-dimensionallyvarying periodicity that is essentially perpendicular to a direction ofpropagation of excitation light in-coupled into the opticallytransparent layer (a) or of surface plasmon resonance generated in themetal layer. Such special embodiments, for example of opticalwaveguides, are described in WO 92/19976 and in WO 98/09156, where theyare also termed “integrated-optical light pointers”. An advantage ofthis embodiment of a grating coupler is based on the fact that an outervariable of a resonance angle for in-coupling an excitation lightdelivered to in-coupling grating from outside is converted into a localvariable on grating-waveguide structure, i.e. into a determination of aposition on this structure, on which a resonance condition is met basedon a suitable period of this coupling grating.

For manufacture of multi-diffractive grating structures or of gratingstructures with three-dimensionally variable periodicity or othercomplex grating structures in the waveguiding layer (a) (or the metallayer, respectively) in relatively large quantities, principally planaroptical structures with plastic substrates (as essentially opticallytransparent layer (b)) are more suitable than such with glasssubstrates, because a manufacturing process of such complex gratingstructures is extremely tedious. It may for example be performed usingmultiple exposure by photolithographic structure, or by structuringusing an electron beam process. Accordingly, manufacture of a master,for example with a glass substrate, is very costly. From such a master,however, it is then possible to mold almost as many copies as one wishesof planar optical structures based on plastic substrates.

For most applications, it is desirable to in-couple excitation lightfrom a spectrum between near-UV and near-IR, i.e. predominantly from avisible spectrum. For this it is an advantage if the grating structures(c) and if applicable also any additional grating structures (c′) thatare present, show a period of 200 nm-1000 nm.

For most applications, it is further desirable if coupling conditionsare very precisely defined over areas as large as possible and change aslittle as possible. It is therefore preferred for these applications ifa resonance angle for in-coupling and out-coupling of a monochromaticexcitation light or for excitation of a surface plasmon does not vary bymore than 0.1° (as deviation from a mean value) within an area of agrating structure of at least 4 mm² (with sides arranged parallel or notparallel with lines of the grating structure (c)) or over a distance ofat least 2 mm parallel with the lines.

The grating structures (c) and/or (c′) may be relief gratings with anyprofile, for example a rectangular, triangular or semicircular profile.

Preferred embodiments of the planar optical structure according to theinvention for generating an evanescent-field measuring platform comprisethe grating structures (c) and/or (c′) being formed as a relief gratingin the surface of layer (b) facing layer (a) or the metal layer, andbeing transmitted in the manufacturing process of the waveguide at leastto the surface (layer interface) of layer (a) or the metal layer facinglayer (b). Relief gratings formed in the surface of layer (b) facinglayers to be deposited later, layer (a) or the metal layer, aretransmitted to surfaces of not only one, but of several layers when theyare deposited on layer (b).

For analytical applications, a general preference is for embodiments ofa planar optical film waveguide according to the invention whichcomprise biological or biochemical or synthetic recognition elementsbeing deposited on the surface of layer (a) or the metal layer,respectively, or on an adhesion-promoting layer additionally depositedon layer (a) or the metal layer for qualitative and/or quantitativedetection of at least one analyte in at least one sample brought intocontact with the recognition elements.

There are numerous methods for depositing the biological or biochemicalor synthetic recognition elements on the optically transparent layer (a)or metal layer. For example, this may take place through physicaladsorption or electrostatic interaction. Orientation of the recognitionelements is then generally statistical. There is also a risk that, ifthere is a difference in composition of a sample containing an analyteor reagents used in a detection method, some of immobilized recognitionelements will be washed away. It may therefore be an advantage if, forimmobilization of biological or biochemical or synthetic recognitionelements (e), an adhesion-promoting layer (f) is deposited on theoptically transparent layer (a) or the metal layer. Thisadhesion-promoting layer should be essentially optically transparent. Inparticular, the adhesion-promoting layer should not jut out fromwaveguiding layer (a) or the metal layer beyond a penetration depth ofthe evanescent field into medium above. The adhesion-promoting layer (f)should therefore have a thickness of less than 200 nm, preferably ofless than 20 nm. This layer may comprise, for example, chemicalcompounds from a group of silanes, functionalized silanes, epoxides,functionalized, charged or polar polymers, thiols, dextrans and“self-assembled passive or functionalized monolayers or multilayers”.

To enable simultaneous detection of multiple and generally differentanalytes, it is preferred if the biological or biochemical or syntheticrecognition elements are immobilized in discrete (spatially separated)measurement areas.

Within a meaning of the present invention, discrete (spatiallyseparated) measurement areas shall be defined by an area which take upbiological or biochemical or synthetic recognition elements immobilizedthere for recognition of at least one analyte in a liquid sample. Theseareas may be present in any geometric form, for example in the form ofpoints, circles, rectangles, triangles, ellipses or stripes. Thereby, itis possible in this case to generate spatially separated measurementareas by spatially selective deposition of biological or biochemical orsynthetic recognition elements on the optical film waveguide (eitherdirectly on the waveguiding layer (a) or the metal layer, respectively,or on an adhesion-promoting layer deposited on layer (a) or the metallayer, respectively). In contact with an analyte or an analog of theanalyte that competes with the analyte for binding to immobilizedrecognition elements or a further binding partner in a multistep assay,these molecules bind only selectively to a surface of the planar opticalstructure in the measurement areas, which are defined by areas which areoccupied by the immobilized recognition elements. It is possible that,in a 2-dimensional arrangement, up to 1,000,000 measurement areas may bearranged on a planar optical structure according to the invention forgenerating an evanescent-field measuring platform, where a singlemeasurement area for example may occupy an area of 0.001 mm²-6 mm².Typically, a density of measurement areas may be more than 10,preferably more than 100, especially preferably more than 1000measurement areas per square centimeter on the surface of layer (a) orthe metal layer, respectively, or on an adhesion-promoting layeradditionally deposited on layer (a) or the metal layer, respectively.

For spatially selective deposition of the biological or biochemical orsynthetic recognition elements, at least one method may be used from agroup of methods comprising “ink jet spotting”, mechanical spotting byuse of a pin, pen or capillary, “micro contact printing”, fluidiccontact of measurement areas with the biological or biochemical orsynthetic recognition elements through their application parallel to orintersecting microchannels, upon exposure to pressure differences or toelectric or electromagnetic potentials, and photochemical orphotolithographic immobilization methods.

For industrial use of a planar optical structure according to theinvention for generating an evanescent-field measuring platform, it isan advantage if established laboratory robots can be used for depositionof the recognition elements and/or samples. This possibility is given ifdimensions of the optical structure are compatible with dimensions ofindustrial standard microtiter plates. Such plates with 96 wells(recesses as sample compartments) spaced at about 9 mm, 384 wells spacedat about 4.5 mm or 1536 wells spaced at about 2.25 mm are commerciallyavailable. It is therefore preferred if outer measurements of a bottomsurface of a planar optical structure according to the invention forgenerating an evanescent-field measuring platform match a footprint ofstandard microtiter plates of about 8 cm×12 cm (with 96 or 384 or 1536wells).

The fact that a substrate (essentially optically transparent layer (b))of a planar optical structure according to the invention for generatingan evanescent-field measuring platform can be structured by molding froma suitable master creates a possibility of also structuring recesses inthis substrate at the same time, before subsequent deposition of thewaveguiding layer (a) or the metal layer, with molding of gratingstructures (c) or (c′) for generating sample compartments in which,after deposition of further layers, discrete (spatially separated)measurement areas are then created with the recognition elementsimmobilized therein. A preferred embodiment of a planar opticalstructure according to the invention for generating an evanescent-fieldmeasuring platform therefore comprises recesses being structured inlayer (b) to create sample compartments. These recesses preferably havea depth of 20 μm to 500 μm, especially preferably 50 μm to 300 μm.

It is also an advantage if the planar optical structure for generatingan evanescent-field measuring platform comprises mechanically and/oroptically recognizable markings to facilitate their adjustment in anoptical system and/or to facilitate a combination of the planar opticalstructure with a further body for creation of at least one samplecompartment.

As biological or biochemical or synthetic recognition elements,components may be applied from a group formed from nucleic acids (forexample DNA, RNA, oligonucleotides) and nucleic acid analogs (e.g. PNA)and derivatives thereof with synthetic bases, monoclonal or polyclonalantibodies and antibody fragments, peptides, enzymes, aptamers,synthetic peptide structures, glycopeptides, oligosaccharides, lectins,soluble, membrane-bound proteins and proteins isolated from a membranesuch as receptors, ligands thereof, antigens for antibodies (e.g. biotinfor streptavidin), “histidine tag components” and complexing partnersthereof, and cavities created by chemical synthesis for accommodatingmolecular imprints, and the like.

The last-named type of recognition elements are understood to meancavities which are manufactured in a process that has been described inliterature as “molecular imprinting”. To this end, an analyte or ananalog of the analyte is encapsulated in a polymer structure, usually inorganic solution. This is then described as an “imprint”. The analyte orthe analog thereof is then removed again from the polymer structure withaddition of suitable reagents, so that it leaves behind an empty cavity.This empty cavity can then be used as a binding site with high stericselectivity in a later detection method.

It is also possible that whole cells or cell fragments may be depositedas biochemical or biological recognition elements.

In many cases, a limit of detection of an analytical method is limitedby signals of so-called nonspecific binding, i.e. by signals that aregenerated by binding of an analyte or other compounds used for detectionof the analyte, which are bound, for example through hydrophobicadsorption or electrostatic interactions, not only in a region ofimmobilized biological or biochemical or synthetic recognition elementsused, but also in areas on a surface of a planar optical structure thatare not covered by these elements. It is therefore an advantage if areasbetween discrete measurement areas are “passivated” to minimizenonspecific binding of analytes or their tracer substances, i.e. ifcompounds “chemically neutral” to the analyte or one of its tracersubstances are deposited between the discrete measurement areas.“Chemically neutral” compounds are understood to be those substanceswhich do not themselves show any specific binding sites for recognitionand binding of the analyte or an analog thereof or a further bindingpartner in a multistep assay and which, through their presence, blockaccess of the analyte or its analog or the further binding partners to asurface of the film waveguide.

“Chemically neutral” compounds which may be used, for example, aresubstances from groups comprising albumins, especially bovine serumalbumin or human serum albumin, casein, nonspecific, polyclonal ormonoclonal, heterologous or for an analyte or analytes to be determinedempirically nonspecific antibodies (especially for immunoassays),detergents (such as Tween 20), fragmented natural or synthetic DNA nothybridizing with polynucleotides for analysis, such as a herring orsalmon sperm extract (especially for polynucleotide hybridizationassays), or also uncharged, but hydrophilic polymers, such aspolyethylene glycols or dextrans.

Especially selection of these substances for reducing nonspecifichybridization in polynucleotide hybridization assays (such as extractsof herring or salmon sperm) is determined here by empirical preferencefor DNA which is “heterologous” for polynucleotides to be analyzed,about which no interactions with polynucleotide sequences to be detectedare known.

A further subject of the invention is an analytical system with a planaroptical structure for generating an evanescent-field measuring platform,with biological or biochemical or synthetic recognition elementsimmobilized on surface of layer (a) or a metal layer, respectively, oron an adhesion-promoting layer additionally deposited on layer (a) orthe metal layer, respectively, for qualitative and/or quantitativedetection of at least one analyte in at least one sample brought intocontact with the recognition elements, wherein an upper side of theplanar optical structure with measurement areas over the opticallytransparent layer (a) or the metal layer created thereon is combinedwith a further body such that between the planar optical structure as abaseplate and the body at least one cavity is formed for creation of atleast one sample compartment, fluidically sealed against one other, ineach of which are located at least one measurement area or segment orarray of measurement areas.

A one-dimensional or two-dimensional arrangement of measurement areaswhich together are brought into contact with the same sample shall bedescribed here as an array of measurement areas. Within a samplecompartment, there may be one or also more arrays of measurement arrays.An arrangement of at least two measurement areas to which a commonfunction is assigned based on selection of recognition elementsimmobilized therein, for example for referencing or for calibration orfor detection of identical analytes, shall be described as a segment ofmeasurement areas. Segments of measurement areas may be parts of anarray of measurement areas.

A preferred embodiment of an analytical system according to theinvention comprises sample compartments being formed as flow cellsfluidically sealed against one another with at least one inlet and atleast one outlet in each case, and optionally at least one outlet ofeach flow cell in addition leading to a reservoir fluidically connectedto this flow cell to receive fluid exiting the flow cell.

Another possible embodiment comprises sample compartments being open onthat side of a body combined with a planar optical structure as abaseplate which lies opposite measurement areas.

An arrangement of sample compartments of an analytical system accordingto the invention may comprise 2-2000, preferably 2-400, especiallypreferably 2-100 individual sample compartments. Thereby, it ispreferred if a pitch (geometrical arrangement in rows and/or columns) ofthe sample compartments matches a pitch of wells of a standardmicrotiter plate. The sample compartments may have the same or differentcapacities of 0.1 nl-100 μl in each case.

The analytical system according to the invention preferably alsocomprises supply facilities for bringing the at least one sample intocontact with immobilized biological or biochemical or syntheticrecognition elements.

Such embodiments of an analytical system according to the inventionpreferably comprise in addition at least one excitation light source fordelivery of at least one excitation light beam to a planar opticalstructure for generating an evanescent-field measuring platform,according to one of the aforementioned embodiments, and at least onedetector for detecting light emanating from the optical structure.

Various methods can be distinguished for analyte detection in anevanescent field of guided light waves in optical film waveguides or inan evanescent field of surface plasmons generated in metal films. Onbasis of a measurement principle used, for example, a distinction can bedrawn between fluorescence or, more generally, luminescence methods onthe one hand and refractive methods on the other. Methods for generatinga surface plasmon resonance in a thin metal layer on a dielectric layerwith lower refractive index can be included here in a group ofrefractive methods, provided a resonance angle of delivered excitationlight for generating surface plasmon resonance is used as a basis fordetermining a parameter. The surface plasmon resonance may also be used,however, to intensify a luminescence or to improve asignal-to-background ratio in a luminescence measurement. Conditions forgenerating a surface plasmon resonance and for combination withluminescence measurements, as well as with waveguiding structures, arewidely described in literature, for example in U.S. Pat. Nos. 5,478,755,5,841,143, 5,006,716 and 4,649,280.

In this application, the term “luminescence” describes spontaneousemission of photons in a range from ultraviolet to infrared, afteroptical or non-optical excitation, such as electrical or chemical orbiochemical or thermal excitation. For example, chemiluminescence,bioluminescence, electroluminescence, and especially fluorescence andphosphorescence are included under the term “luminescence”.

In refractive methods of measurement, a change in the so-calledeffective refractive index resulting from molecular adsorption ordesorption on a waveguide is used for detection of the analyte. Thischange in the effective refractive index, in a case of grating couplersensors, is determined for example from a change in a coupling angle forin-coupling or out-coupling of light into or out of a grating couplersensor, and in a case of interferometric sensors it is determined from achange in a phase difference between measuring light guided in a sensorarm and a reference arm of an interferometer. In a case of surfaceplasmon resonance, for example, a corresponding change in the resonanceangle for generating a surface plasmon is measured. Provided anexcitation light source used is tunable over a certain spectral range,changes in an excitation wavelength at which in-coupling into a gratingcoupler sensor or excitation of a surface plasmon occur can also bemeasured, instead of change in the coupling or resonance angle,respectively, with a fixed angle of incidence. The refractive methodsmentioned have an advantage in that they can be employed without use ofadditional marker molecules, so-called molecular labels. However, theyare generally less sensitive than detection methods which are based ondetermination of luminescence excited in an evanescent field of awaveguide.

In a case of refractive measurement techniques, detection of measurementlight typically takes place at a wavelength of excitation light. Acharacteristic of embodiments of the analytical system according to theinvention which are particularly suitable for refractive methods ofdetection is therefore that detection of light at a wavelength ofirradiated excitation light and emanating from a planar opticalstructure, for generating an evanescent-field measuring platform as partof the analytical system, is performed.

For known arrangements of grating coupler sensors (see e.g.: K.Tiefenthaler, W. Lukosz, “Sensitivity of grating couplers asintegrated-optical chemical sensors”, J. Opt. Soc. Am. B6, 209 (1989);W. Lukosz, Ph. M. Nellen, Ch. Stamm, P. Weiss, “Output Grating Couplerson Planar Waveguides as Integrated, Optical Chemical Sensors”, Sensorsand Actuators B1, 585 (1990), and in T. Tamir, S. T. Peng, “Analysis andDesign of Grating Couplers”, Appl. Phys. 14, 235-254 (1977)) a locallyresolved measurement is not possible. PCT/EP 01/00605 contains adescription of a grating-waveguide structure which enables changes inresonance conditions for in-coupling of excitation light intowaveguiding layer (a) of an optical film waveguide via a gratingstructure (c) modulated in layer (a), or for out-coupling of lightguided in layer (a), with arrays of measurement areas generated thereon,each with different immobilized biological or biochemical or syntheticrecognition elements for simultaneous binding and detection of at leastone analyte, to be measured in a locally resolved manner, whereexcitation light is delivered at the same time to an entire array ofmeasurement areas, and a degree of fulfillment of a resonance conditionfor in-coupling of light into layer (a) towards the measurement areas ismeasured at the same time. Planar optical structures on which thepresent invention is based for generating an evanescent-field measuringarrangement, especially in the embodiment of planar optical filmwaveguides, are suitable for such imaging refractive methods ofmeasurement. Embodiments of planar film waveguides described in PCT/EP01/00605, as grating-waveguide structures, as well as likewise describedoptical systems, as an integral part of corresponding embodiments ofanalytical systems according to the invention, as well as methods basedon use thereof for detection of at least one analyte, are thereforelikewise a subject of the present invention.

Other embodiments of an analytical system according to the invention arecharacterized in that detection of light emanating from the planaroptical structure is performed at a wavelength other than that ofincident excitation light. It is preferred if detection of lightemanating from the planar optical structure is performed at a wavelengthof a luminescence excited by the excitation light. Thereby, a wavelengthof detected luminescence is generally shifted to wavelengths longer thanthat of the incident excitation light.

For the in-coupling of excitation light into optically transparent layer(a) of a planar optical film waveguide, an analytical system accordingto the invention may comprise at least one optical in-coupling elementfrom a group comprising prism couplers, evanescent couplers withcombined optical waveguides with overlapping evanescent fields, butt-endcouplers with focusing lenses, preferably cylinder lenses, arranged infront of one face of a waveguiding layer, and grating couplers. It ispreferred if excitation light is in-coupled into the opticallytransparent layer (a) using at least one grating structure (c) featuredin the optically transparent layer (a), where the excitation light isdelivered to the at least one grating structure featured in layer (a)under an angular range which comprises a resonance angle for in-couplinginto layer (a) via the at least one grating structure. Accordingly, tomeet resonance conditions for generating a surface plasmon in a metallayer of a planar optical structure according to the invention, it ispreferred if excitation of the surface plasmon resonance takes placeusing at least one grating structure (c) featured in the metal layer,where the excitation light is delivered to the at least one gratingstructure featured in the metal layer under an angular range whichcomprises the resonance angle.

In this case, it is preferable if an incident excitation light in eachcase is essentially parallel and monochromatic and is delivered to theat least one grating structure featured in layer (a) or (a′) under theresonance angle for in-coupling into layer (a) or for excitation of thesurface plasmon in the metal layer.

Light guided in an essentially optically transparent layer (a) may beout-coupled using grating structures (c′) which are featured in layer(a).

It is preferred if an analytical system according to the inventioncomprises in addition at least one positioning element for changing anangle of an incident excitation light between its direction ofpropagation in free space, characterized by a corresponding k-vector,prior to impingement on a surface of a planar optical structure, forgenerating an evanescent-field measuring platform, and projectionthereof into a plane of the surface of the optical structure. Ananalytical system according to the invention preferably also comprisesat least one positioning element for lateral modification of a site ofimpingement of an incident excitation light on the planar opticalstructure.

A special embodiment of an analytical system according to the inventioncomprises at least one expansion optics with which excitation light fromat least one light source is expanded in one direction, if adequatelyparallel with grating lines of a grating structure featured in layer (a)for in-coupling of excitation light into layer (a) and/or in two spatialdirections, if adequately parallel and perpendicular to grating lines ofa grating structure featured in layer (a). It is preferred here if adiameter of an incident excitation light bundle on the planar opticalfilm waveguide at least in one direction in the plane of the surface ofthe waveguide is at least 2 mm, preferably at least 5 mm. Theselast-named special embodiments are especially well-suited for example torealization of an arrangement of an imaging grating coupler, asdescribed in PCT/EP 01/00605 and fully included in this invention.

It is preferred if an analytical system according to the inventioncomprises at least one locally resolved detector, which is preferablyselected from a group of detectors comprising CCD cameras, CCD chips,photodiode arrays, avalanche diode arrays, multichannel plates andmultichannel photomultipliers.

Preference is also for embodiments of an analytical system according tothe invention which comprise use of optical components from a groupcomprising lenses or lens systems for forming transmitted light bundles,planar or curved mirrors for deflection and if necessary additionallyfor forming light bundles, prisms for deflecting and if necessary forspectral division of light bundles, dichroic mirrors for spectrallyselective deflection of parts of light bundles, neutral filters forregulation of transmitted light intensity, optical filters ormonochromators for spectrally selective transmission of parts of lightbundles or polarization-selective elements for selection of discretepolarization directions of excitation or luminescence light, with thesecomponents being arranged between the at least one excitation lightsource and the planar optical structure according to the invention forgenerating an evanescent-field measuring platform and/or between theplanar optical structure and the at least one detector.

It is possible that excitation light is delivered in pulses with aduration between 1 fsec and 10 minutes, and light emanating from theplanar optical structure is measured in a time-resolved manner. Inparticular, with such an embodiment binding of at least one analyte torecognition elements in various measurement areas can be observed inreal time in a locally resolved manner. Respective binding kinetics canbe determined from signals recorded in time-resolved measurements. Thisallows in particular, for example, a comparison of affinities ofdifferent ligands to an immobilized biological or biochemical orsynthetic recognition element. In this context, any binding partner ofsuch an immobilized recognition element shall be described as a“ligand”.

It is preferred if an analytical system according to the inventioncomprises arrangements which allow (1) measurement of essentiallyisotropically emitted light from a planar optical film waveguideaccording to the invention, and optionally measurement areas locatedthereon or (2) light back-coupled into the optically transparent layerand outcoupled via grating structures featured in layer (a) or light ofboth parts (1) and (2).

A special embodiment of an analytical system according to the inventioncomprises arrangements which allow delivery of excitation light anddetection of light emanating from at least one measurement area to takeplace sequentially for single or several measurement areas.

These arrangements may consist in sequential excitation and detectionusing movable optical components from a group comprising mirrors,deflecting prisms and dichroic mirrors.

An integral part of the invention is also a system wherein sequentialexcitation and detection take place using an essentially angle- andfocus-preserving scanner. It is also possible that the planar opticalstructure is moved between steps of sequential excitation and detection.

A further subject of the invention is a method for qualitative and/orquantitative detection of at least one analyte in at least one sample,wherein the at least one sample is brought into contact with biologicalor biochemical or synthetic recognition elements, which are immobilizeddirectly or indirectly, via an adhesion-promoting layer, on a surface ofa planar optical structure for generating an evanescent-field measuringplatform, according to the invention and any of the embodiments, andwherein changes for in-coupling of an incident excitation light in awaveguiding film (a) of a planar optical film waveguide and/or forout-coupling of light emanating from the film waveguide or forgenerating a surface plasmon in a metal layer, as a result of thebinding of the at least one analyte or of one of binding partnersthereof to at least one immobilized recognition element, is measured.

It is preferred if the biological or biochemical or syntheticrecognition elements are immobilized in discrete measurement areas.

It is also preferred if the in-coupling of excitation light into thewaveguiding layer (a) or the generation of a surface plasmon in themetal layer takes place using at least one grating structure (c)featured in layer (a) or the metal layer, respectively.

Certain embodiments of the process according to the invention comprisedetection of at least one analyte being performed on basis of changes inan effective refractive index, as a result of binding of this analyte,and where applicable of one of its binding partners, to biological orbiochemical or synthetic recognition elements immobilized on a gratingstructure featured in layer (a) or the metal layer, respectively, and onbasis of resulting changes in resonance conditions for in-couplingexcitation light into layer (a) or for generating a surface plasmon inthe metal layer using the grating structure.

A characteristic of other embodiments of the method is that at least oneanalyte is detected on basis of changes in conditions for out-coupling alight guided in layer (a) via a grating structure (c) or (c′) featuredin layer (a), as a result of binding of this analyte, and whereapplicable of any of its binding partners, to biological or biochemicalor synthetic recognition elements immobilized on the grating structure,and of associated changes in the effective refractive index.

A further preferred subject of the invention is a method for qualitativeand/or quantitative detection of at least one analyte in at least onesample, wherein this sample is brought into contact with biological orbiochemical or synthetic recognition elements immobilized directly orindirectly, via an adhesion-promoting layer, on a surface of a planaroptical structure according to the invention for generating anevanescent-field measuring platform in accordance with one of theembodiments, wherein excitation light from at least one light source isin-coupled into layer (a) and guided therein, and wherein luminescenceof molecules capable of luminescence, which are bound to the analyte orto one of its binding partners, is excited and measured in a near-fieldof layer (a).

The second essentially optically transparent layer (b) here preferablycomprises a material from a group formed by cyclo-olefin polymers andcyclo-olefin copolymers.

This method according to the invention enables (1) isotropically emittedluminescence or (2) luminescence in-coupled into the opticallytransparent layer (a) and out-coupled via grating structure (c) or (c′),or luminescences of both (1) and (2), to be measured simultaneously.

For generation of luminescence, it is preferred if a luminescence dye orluminescent nanoparticle is used as a luminescence label, which can beexcited and emits at a wavelength between 300 nm and 1100 nm.

The luminescence label may be bound to the analyte or, in a competitiveassay, to an analog of the analyte or, in a multistep assay, to one ofbinding partners of immobilized biological or biochemical or syntheticrecognition elements or to the biological or biochemical or syntheticrecognition elements.

A characteristic of special embodiments of the method according to theinvention comprises use of a second luminescence label or furtherluminescence labels with excitation wavelengths either the same as ordifferent from that of the first luminescence label and the same ordifferent emission wavelength. Through appropriate selection of spectralcharacteristics of luminescence labels used, such embodiments may bedesigned such that the second luminescence label or further luminescencelabels can be excited at the same wavelength as the first luminescencelabel, but may emit at a different wavelength.

For certain applications, for example for measurements independent ofone another with different excitation and detection labels, it is anadvantage if excitation spectra and emission spectra of luminescencedyes show little, if any, overlap.

Another special embodiment of the method comprises using charge oroptical energy transfer from a first luminescence dye serving as donorto a second luminescence dye serving as acceptor for a purpose ofdetecting an analyte.

A characteristic of another special embodiment of the method accordingto the invention comprises determining changes in an effectiverefractive index on measurement areas in addition to measuring at leastone luminescence.

It is an advantage if the at least one luminescence and/ordeterminations of light signals at an excitation wavelength areperformed in a polarization-selective manner. In particular, it ispreferred if the at least one luminescence is measured at a polarizationdifferent from the one of the excitation light.

The method according to the invention comprises samples to be testedbeing aqueous solutions, in particular buffer solutions or naturallyoccurring body fluids such as blood, serum, plasma, urine or tissuefluids. A sample to be tested may also be an optically turbid fluid,surface water, a soil or plant extract, or a biological or syntheticprocess broth. The samples to be tested may also be prepared frombiological tissue parts or cells.

A further subject of the invention is use of a planar optical structureaccording to the invention for generating an evanescent-field measuringplatform according to one of the aforementioned embodiments and/or ananalytical system according to the invention and/or a method accordingto the invention for detection of at least one or more analyte forquantitative and/or qualitative analyses to determine chemical,biochemical or biological analytes in screening methods inpharmaceutical research, combinatorial chemistry, clinical andpre-clinical development, for real-time binding studies and to determinekinetic parameters in affinity screening and in research, forqualitative and quantitative analyte determinations, especially for DNAand RNA analytics, for generation of toxicity studies and determinationof gene or protein expression profiles, and for determination ofantibodies, antigens, pathogens or bacteria in pharmaceutical productresearch and development, human and veterinary diagnostics, agrochemicalproduct research and development, for symptomatic and pre-symptomaticplant diagnostics, for patient stratification in pharmaceutical productdevelopment and for therapeutic drug selection, for determination ofpathogens, nocuous agents and germs, especially of salmonella, prions,viruses and bacteria, in food and environmental analytics.

The invention is illustrated in the following examples of embodiments.

EXAMPLES 1. Master

A master is obtained from a planar thin-film waveguide (“chip”),comprising a substrate of AF 45 glass, with dimensions 16 mm×48 mm×0.7mm, and a coating of tantalum pentoxide (150 nm thick) as waveguidinglayer. Parallel with a short side of the “chip”, relief gratings spaced9 mm apart are formed for in-coupling of excitation light into thewaveguiding layer, with these gratings having a period of 320 nm, adepth of 12 nm and a length (in a direction of propagation of guidedlight, i.e. parallel with a longitudinal side of the “chip”) of 0.5 mm.

A piece measuring 12 mm×25 mm is cut out of the “chip” such that threegratings are formed thereon with grating lines parallel with a shortside of the piece.

2. Process For Manufacture of A Body From A ThermoplasticPlastic/Process For Manufacture of A Planar Optical Film WaveguideAccording To the Invention

Molds of the master described under 1. are manufactured in the followingplastics: 1. Polycarbonate (PC), 2. Polymethylmethacrylate (PMMA), 3.Cyclo-olefin copolymer (COC), 4. Cyclo-olefin polymer (COP).

The master is inserted into a molding tool. Molding is performedaccording to the variotherm injection molding process (A. Rogalla,“Analyse des Spritzgiessens mikrostrukturierter Bauteile ausThermoplasten”, IKV Berichte aus der Kunststoffverarbeitung, Band 76,Verlag Mainz, Wissenschaftsverlag Aachen, Germany, 1998).

The molding tool with this integrated master is closed, producing acavity therein in the form of a “nest”, and is heated to a meltingtemperature (molding temperature, up to 180° C. for PC, up to 140° C.for PMMA, up to 180° C. for COC and up to 170° C. for COP). An injectionunit then travels as far as the nest, which is evacuated at the sametime to a residual pressure between 10 mbar and 300 mbar.

Plastic is then injected at a pressure of 800 bar-1800 bar in the caseof PC, 600 bar-1200 bar in the case of PMMA, 500 bar-1500 bar in thecase of COC, and 500 bar-1800 bar in the case of COP. After the cavityis filled, heating is switched off, and the unit starts cooling down toa demolding temperature (100° C.-140° C. for PC, 70° C.-100° C. forPMMA, 70° C.-160° C. for COC and 80° C.-140° C. for COP, specific tomolding in each case).

Before an injected plastic phase solidifies, post-molding is performed(600 bar-1200 bar for PC, 400 bar-600 bar for PMMA, 300 bar-500 bar forCOC and 500 bar-1500 bar for COP) to counteract material shrinkage oncooling.

The injection unit then returns, and the cavity (nest) is aerated.

For preparation of a next injection process, new plastic pellets are fedin and plasticized for a next injection. The injection tool is opened,and a molded plastic structure is ejected.

This machine is thus ready for starting a next injection cycle.

1-97. (canceled)
 98. A planar optical structure for generating anevanescent-field measuring platform, wherein said evanescent-fieldmeasuring platform comprises a multilayer system, with a metal layerand/or an essentially optically transparent, waveguiding layer (a) withrefractive index n₁ and at least a second, essentially opticallytransparent layer (b) with refractive index n₂, where n₁>n₂, and wherethe second layer (b) consists of a thermoplastic plastic and a materialfrom the group formed by cyclo-olefin polymers and cyclo-olefincopolymers.
 99. A planar optical structure for generating anevanescent-field measuring platform according to claim 98, wherein saidevanescent-field measuring platform is a planar optical structure forgenerating a surface plasmon resonance.
 100. A planar optical structurefor generating an evanescent-field measuring platform according to claim98, wherein said evanescent-field measuring platform is a planar opticalfilm waveguide, comprising a first essentially optically transparentwaveguiding layer (a) with refractive index n₁ and a second essentiallyoptically transparent layer (b) with refractive index n₂, where n₁>n₂,and where the second layer (b) of said film waveguide comprises amaterial from the group formed by cyclo-olefin polymers and cyclo-olefincopolymers.
 101. A planar optical film waveguide according to claim 100,wherein the refractive index of the first optically transparent layer(a) is greater than 1.8.
 102. A planar optical film waveguide accordingto claim 100, wherein the first optically transparent layer (a)comprises Ti0 ₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂, especially preferablyTiO₂ and Ta₂O₅.
 103. A planar optical film waveguide according to claim100, wherein the waveguiding layer (a) is in optical contact with atleast one optical coupling element for in-coupling of excitation lightof one or more wavelengths, from one or more light sources, into layer(a).
 104. A planar optical film waveguide according to claim 100,wherein light guided in the optically transparent layer (a) isout-coupled using grating structures (c′), which are featured in theoptically transparent layer (a).
 105. A planar optical film waveguideaccording to claim 100, wherein a further optically transparent layer(b′) with a lower refractive index than that of layer (a) and with athickness of 5 nm-10000 nm, preferably 10 nm-1000 nm, is providedbetween the optically transparent layers (a) and (b) and is in contactwith layer (a).
 106. A planar optical structure for generating anevanescent-field measuring platform according to claim 98, whereinlarge-area grating structures (c) and/or (c′) are featured overextensive surface areas of said optical structure, preferably over theentire surface area thereof.
 107. A planar optical structure forgenerating an evanescent-field measuring platform according to claim 98,comprising multiple grating structures (c) and/or (c′) on a common,continuous substrate in the essentially optically transparent layer (a)and/or the metal layer.
 108. A planar optical structure for generatingan evanescent-field measuring platform according to claim 98, comprisinga superposition of 2 or more grating structures of differing periodicitywith a parallel or nonparallel arrangement of the grating lines.
 109. Aplanar optical structure for generating an evanescent-field measuringplatform according to claim 98, wherein one or more grating structures(c) and/or (c′) show a three-dimensionally varying periodicity that isessentially perpendicular to the direction of propagation of theexcitation light in-coupled into the optically transparent layer (a) orof the surface plasmon resonance generated in the metal layer.
 110. Aplanar optical structure for generating an evanescent-field measuringplatform according to claim 98, wherein grating structures (c) and,where applicable, additional grating structures (c′) have a period of200 nm-1000 nm.
 111. A planar optical structure for generating anevanescent-field measuring platform according to claim 98, wherein theresonance angle for in-coupling and out-coupling of a monochromaticexcitation light or for excitation of a surface plasmon within an areaof a grating structure of at least 4 mm² (with the sides arranged inparallel or not parallel with the lines of the grating structure (c)) orover a distance of at least 2 mm in parallel with the lines does notvary by more than 0.1 (as deviation from a mean value).
 112. A planaroptical structure for generating an evanescent-field measuring platformaccording to claim 98, wherein grating structures (c) and/or (c′) arerelief gratings with any profile, for example with a rectangular,triangular or semicircular profile.
 113. A planar optical structure forgenerating an evanescent-field measuring platform according to claim 98,wherein said grating structures (c) and/or (c′) are formed as reliefgratings in the surface of layer (b) facing layer (a) and/or the metallayer and are transferred in the manufacturing process of said waveguideat least to the surface of layer (a) or the metal layer facing layer(b).
 114. A planar optical structure for generating an evanescent-fieldmeasuring platform according to claim 98, wherein biological orbiochemical or synthetic recognition elements are deposited on thesurface of layer (a) or the metal layer, or on an adhesion-promotinglayer additionally deposited on layer (a) or the metal layer, for thequalitative and/or quantitative detection of one or more analytes in oneor more samples brought into contact with said recognition elements.115. A planar optical structure for generating an evanescent-fieldmeasuring platform according to claim 98, wherein the outer dimensionsof its base area match the footprint of standard microtiter plates ofabout 8 cm×12 cm (with 96 or 384 or 1536 wells)
 116. A planar opticalstructure for generating an evanescent-field measuring platformaccording to claim 98, wherein recesses are formed in layer (b) tocreate sample compartments.
 117. A planar optical structure forgenerating an evanescent-field measuring platform according to claim 98,comprising mechanically and/or optically identifiable markings tofacilitate adjustment in an optical system and/or to facilitate theconnection of said planar optical structure to a further body forcreating one or more sample compartments.
 118. A planar opticalstructure for generating an evanescent-field measuring platformaccording to claim 98, wherein the outer dimensions of its base areamatch the footprint of standard microtiter plates of about 8 cm×12 cm(with 96 or 384 or 1536 wells)
 119. Use of a planar optical structurefor generating an evanescent-field measuring platform according to claim98 for quantitative and or qualitative analyses to determine chemical,biochemical or biological analytes in screening methods inpharmaceutical research, combinatorial chemistry, clinical andpre-clinical development, for real-time binding studies and to determinekinetic parameters in affinity screening and in research, forqualitative and quantitative analyte determinations, especially for DNAand RNA analytics, for the generation of toxicity studies and thedetermination of gene or protein expression profiles, and for thedetermination of antibodies, antigens, pathogens or bacteria inpharmaceutical product research and development, human and veterinarydiagnostics, agrochemical product research and development, forsymptomatic and pre-symptomatic plant diagnostics, for patientstratification in pharmaceutical product development and for therapeuticdrug selection, for the determination of pathogens, nocuous agents andgerms, especially of salmonella, prions, viruses and bacteria, in foodand environmental analytics.